This invention concerns the cloning of a novel cDNA sequence encoding a particular subunit of the human GABAA receptor. In addition, the invention relates to a stable cell line capable of expressing said cDNA and to the use of the cell line in a screening technique for the design and development of subtype-specific medicaments.
Gamma-amino butyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system. It mediates fast synaptic inhibition by opening the chloride channel intrinsic to the GABAA receptor. This receptor comprises a multimeric protein of molecular size 230-270 kDa with specific binding sites for a variety of drugs including benzodiazepines, barbiturates and xcex4-carbolines, in addition to sites for the agonist ligand GABA (for reviews see Stephenson, Biochem. J., 1988, 249, 21; Olsen and Tobin, Faseb J., 1990, 4, 1469; and Sieghart, Trends in Pharmacol. Sci., 1989, 10, 407).
Molecular biological studies demonstrate that the receptor is composed of several distinct types of subunit, which are divided into four classes (xcex1, xcex2, xcex3 and xcex4) based on their sequence similarities. To date, six types of xcex1 (Schofield et al., Nature (London), 1987, 328, 221; Levitan et al., Nature (London), 1988, 335, 76; Ymer et al., EMBO J., 1989, 8, 1665; Pritchett and Seeberg, J. Neurochem., 1990, 54, 802; Luddens et al., Nature (London), 1990, 346, 648; and Khrestchatisky et al., Neuron, 1989, 3, 745), three types of xcex2 (Ymer et al., EMBO J., 1989, 8, 1665), three types of y (Ymer et al., EMBO J., 1990, 9, 3261; Shivers et al., Neuron, 1989, 3, 327; and Knoflach et al, FEBS Lett., 1991, 293, 191) and one 6 subunit (Shivers et al., Neuron, 1989, 3, 327) have been identified.
The differential distribution of many of the subunits has been characterised by in situ hybridisation (Sequier et al., Proc. Natl. Acad. Sci. USA, 1988, 85, 7815; Malherbe et al., J. Neurosci., 1990, 10, 2330; Shivers et al., Neuron, 1989, 3, 327; and Wisden et al, J. Neurosci., 1992, 12, 1040) and this has permitted it to be speculated which subunits, by their co-localisation, could theoretically exist in the same receptor complex.
Various combinations of subunits have been co-transfected into cells to identify synthetic combinations of subunits whose pharmacology parallels that of bona fide GABAA receptors in vivo (Pritchett et al., Science, 1989, 245, 1389; Malherbe et al., J. Neurosci., 1990, 10, 2330; Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802; and Luddens et al., Nature (London), 1990, 346, 648). This approach has revealed that, in addition to an xcex1 and xcex2 subunit, either xcex31 or xcex32 (Pritchett et al. Nature (London), 1989, 338, 582; Ymer et al., EMBO J., 1990, 9, 3261; and Malherbe et al., J. Neurosci., 1990, 10, 2330) or y3 (Herb et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 1433; Knoflach et al., FEBS Lett., 1991, 293, 191; and Wilson-Shaw et al., FEBS Lett., 1991, 284, 2 11) is also generally required to confer benzodiazepine sensitivity, and that the benzodiazepine pharmacology of the expressed receptor is largely dependent on the identity of the xcex1 and xcex3 subunits present. Receptors containing a xcex4 subunit (i.e. xcex1xcex2xcex4) do not appear to bind benzodiazepines (Shivers et al., Neuron, 1989, 3, 327). Combinations of subunits have been identified which exhibit the pharmacological profile of a BZ1 type receptor (xcex11xcex21xcex32) and a BZ2 type receptor (xcex12xcex21xcex32 or xcex13xcex21xcex32, Pritchett et al., Nature (London), 1989, 338, 582), as well as two GABAA receptors with a novel pharmacology, xcex15xcex22xcex32 (Pritchett and Seeberg, J. Neurochem., 1990, 54, 1802) and xcex16xcex22xcex32 (Luddens et al., Nature (London), 1990, 346, 648). Although the pharmacology of these expressed receptors appears similar to that of those identified in brain tissue by radioligand binding, it has nonetheless not been shown that these receptor subunit combinations exist in vivo.
A combination of subunits comprising either the human xcex14 GABAA receptor subunit and/or the xcex4 GABAA receptor subunit has not hitherto been possible due to the non-availability of the human xcex14 cDNA or human xcex4 cDNA. This has consequently limited the use of cell lines in screening for subtype-specific medicaments, it being impossible to study the pharmacological profile of subunit combinations comprising the xcex14 subunit and/or the xcex4 subunit.
We have now ascertained the cDNA sequence of the xcex14 subunit and the xcex4 subunit of the human GABAA receptor. These nucleotide sequences sequence (SEQ ID NO:7 and SEQ ID NO:11), together with their deduced amino acid sequences sequence (SEQ ID NO:8 and SEQ ID NO:12) corresponding thereto, are depicted in FIGS. 2 and 3 of the accompanying drawings.
The present invention accordingly provides, in a first aspect, a DNA molecule encoding the xcex14 subunit of the human GABAA receptor comprising all or a portion of the sequence (SEQ ID NO:7) depicted in FIG. 2, or a modified human sequence.
The present invention also provides, in another aspect, a DNA molecule encoding the xcex4 subunit of the human GABAA receptor comprising all or a portion of the sequence (SEQ ID NO:11) depicted in FIG. 3, or a modified human sequence.
The sequencing of the novel cDNA molecules in accordance with the invention can conveniently be carried out by the standard procedure described in accompanying Example 1; or may be accomplished by alternative molecular cloning techniques which are well known in the art, such as those described by Maniatis et al. in Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York, 2nd edition, 1989.
In another aspect, the invention provides a recombinant expression vector comprising the nucleotide sequence of the human GABAA receptor xcex14 subunit together with additional sequences capable of directing the synthesis of the said human GABAA receptor xcex14 subunit in cultures of stably co-transfected eukaryotic cells.
The present invention also provides a recombinant expression vector comprising the nucleotide sequence of the human GABAA receptor xcex4 subunit together with additional sequences capable of directing the synthesis of the said human GABAA receptor xcex4 subunit in cultures of stably co-transfected eukaryotic cells.